Hippo信号通路在LPS诱导的肺癌A549细胞增殖的机制研究

发布时间：2018-04-21 06:50

本文选题：LPS + Hippo信号通路　；参考：《扬州大学》2017年硕士论文

【摘要】：目的:炎症已经被认为是癌症的第七种生物学特性。很多肺部慢性炎症也能够增加肺癌发生的风险,并在一定程度上促进肺癌的进展。脂多糖(Lipopolysaccharides,LPS)是革兰氏阴性杆菌释放的重要致炎因子,具有引起炎性免疫应答及内毒素休克等效应。一些既往研究显示,Hippo信号通路参与了肺癌细胞的增殖。本研究拟探究Hippo信号通路在LPS诱导的肺腺癌A549细胞增殖过程中的作用及其机制。方法:1、培养A549细胞株,应用CCK-8检测LPS对A549增殖能力的影响;2、应用Western blot(WB)分别检测不同浓度LPS处理A549后Hippo信号通路相关蛋白(Mst1、SAV1、LATS1、MOB1、P-MOB1、YAP 和 P-YAP(ser127))的表达变化;应用WB分别检测LPS处理不同时间点A549中Hippo信号通路相关蛋白(Mst1、YAP、P-YAP(ser127))的表达变化;应用免疫荧光检测LPS处理后A549中YAP蛋白的定位变化;3、A549转染Cy3标记的siRNA,应用免疫荧光显微镜观察转染效率;应用WB、逆转录-多聚酶链反应(Reverse transcription polymerase chain reaction,RT-PCR)检测目的蛋白和mRNA的抑制率;4、A549转染 Mst1 siRNA、YAPsiRNA 后,抑制 Mst1、YAP 表达,并应用 CCK-8检测LPS对转染Mst1 siRNA、YAP siRNA后A549增殖能力的变化;5、A549转染Mst1siRNA、YAPsiRNA后,抑制 Mst1、YAP表达,应用 WB检测Mst1、YAP的表达变化;应用免疫荧光技术检测YAP蛋白定位变化。结果:1、0.1 μg/ml的LPS可显著促进A549细胞增殖;2、LPS能够下调A549细胞中Mst1、LATS1和p-mob1总蛋白的表达,上调SAV1、Mob1、YAP和p-YAP总蛋白的表达以及促进YAP的核易位;LPS对肺癌A549细胞中YAP蛋白的定位无明显影响;3、Mst1 siRNA和YAP siRNA能够成功转染至肺癌A549细胞中,并且显著抑制了A549细胞中Mst1和YAP的表达;4、转染Mst1 siRNA后LPS对A549细胞的增殖作用无明显变化,而转染YAP siRNA后LPS对A549细胞的促增殖作用明显抑制;5、转染Mst1 siRNA后,LPS能够进一步减少A549中Mst1的表达,并且显著增加YAP的表达;转染YAP siRNA后,LPS对A549中Mst1的表达无明显影响,而对YAP表达的促进作用明显减弱。6、转染Mst1 siRNA和YAP siRNA后,LPS对于A549细胞中YAP的表达定位无明显影响。结论:1、低浓度LPS能够通过上调YAP表达促进A549细胞的增殖;2、YAP并不完全通过Hippo信号通路参与LPS诱导的A549细胞的增殖。
[Abstract]:Objective: inflammation has been recognized as the seventh biological characteristic of cancer. Many chronic lung inflammation also increases the risk of lung cancer and, to some extent, promotes its progression. Lipopolysaccharide lipopolysaccharide (LPSs) is an important inflammatory factor released by gram-negative bacilli and has the effects of inflammatory immune response and endotoxic shock. Some previous studies have shown that Hippo signaling pathway is involved in the proliferation of lung cancer cells. The purpose of this study was to investigate the role of Hippo signaling pathway in the proliferation of A549 cells induced by LPS and its mechanism. Methods: A549 cell line was cultured with CCK-8. The expression of Hippo signaling pathway related protein (Mst1SAV1, MOB1, P-MOB1OYAP and P-YAPser127A) was detected by Western blotWB. The expression of Hippo signal pathway related protein (Mst1SAV1) and P-YAPser127were detected by Western blotWB. The expression of Hippo signal pathway related protein (Mst1) and P-YAPP ser127A in A549 treated with LPS were detected by WB. The localization of YAP protein in A549 treated with LPS was detected by immunofluorescence. The Cy3 labeled siRNAs were transfected with A549 and the transfection efficiency was observed by immunofluorescence microscope. Reverse transcription-polymerase chain reaction (reverse transcription polymerase chain reverse PCR) was used to detect the inhibition rate of target protein and mRNA. Mst1 siRNA-YAPsiRNA was transfected into A549 to inhibit the expression of Mst1 siRNA-YAPsiRNA, and CCK-8 was used to detect the change of proliferation ability of A549 transfected with Mst1 siRNA-YAP siRNA, and 5A1-siRNA-5A549 was transfected into Mst1siRNA-YAPsiRNA. The expression of YAP protein was detected by WB, and the localization of YAP protein was detected by immunofluorescence technique. Results LPS with 0.1 渭 g/ml of 1 渭 g/ml could significantly promote the proliferation of A549 cells. LPs could down-regulate the expression of Mst1, LATS1 and p-mob1 total proteins in A549 cells. Upregulating the expression of total protein of SAV1 and p-YAP and promoting the nuclear translocation of YAP had no significant effect on the localization of YAP protein in lung cancer A549 cells. The expression of Mst1 siRNA and YAP siRNA in lung cancer A549 cells could be successfully transfected into A549 cells. The expression of Mst1 and YAP in A549 cells was significantly inhibited, and the proliferation of A549 cells was not changed after transfection of Mst1 siRNA with LPS. The proliferation of A549 cells was inhibited by LPS after transfection of YAP siRNA, the expression of Mst1 in A549 was further decreased and the expression of YAP was significantly increased after transfection of Mst1 siRNA, and the expression of Mst1 in A549 was not significantly affected by LPS after transfection of YAP siRNA. However, the promotion of YAP expression was significantly weakened. The expression of YAP in A549 cells was not significantly affected by Mst1 siRNA and YAP siRNA transfection. Conclusion low concentration of LPS can promote the proliferation of A549 cells by up-regulating the expression of YAP. (2) Yap does not participate in the proliferation of A549 cells induced by LPS through Hippo signaling pathway.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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